Method for the treatment of diseases or disorders of the inner ear

ABSTRACT

In a process for the treatment of diseases or disorders of the inner ear linked with damage or destruction of sensory cells of the inner ear, for regenerating the sensory cells use is made of at least one active ingredient, which at least partly inhibits or eliminates the inhibiting action of at least one cell cycle inhibitor present in the inner ear. In this process the sensory cells of the inner ear are preferably regenerated by stimulating the proliferation of supporting cells. The sensory cells of the inner ear are so-called hair sensory cells. As cell cycle inhibitors use can be made of cyclin-dependent kinase inhibitors such as in particular the cyclin-dependent kinase inhibitor p27 Kip1 .

This application is a national stage filing of PCT/EP99/01153 filed Feb.23, 1999, and claims benefit of German application 198 07 426.3, filedFeb. 23, 1998.

The invention firstly relates to a process for the treatment of diseasesor disorders of the inner ear, which are linked with damage ordestruction of the sensory cells of the inner ear.

The inner ear of humans and other mammals can either be irreversiblydamaged from the outset by a genetic defect or subsequently by externalinfluences. These external influences can e.g. be acoustic trauma ortoxic or hypoxic influences. Such damage can lead to functionaldisturbances or losses of the senses located in the inner ear,particularly hearing. In the case of these functional disturbancesparticular reference must be made to a reduction or disappearance of thepower of hearing. It is estimated that in Germany approximately 12million people suffer from a so-called perceptive deafness, which can beattributed to the aforementioned pathogenetic mechanisms. Apart from thedegeneration of sensory neurons and damage to the so-called striavascularis of the inner ear, a cause of partial or complete loss of thepower of hearing can in particular be damage or destruction of thesensory cells of the inner ear and consequently the hearing organ.

In a process for the treatment of diseases or disorders of the inner earlinked with damage or destruction of the sensory cells, it must be bornein mind that it is no longer possible to regenerate irreversibly damagedand therefore lost cells in the highly differentiated sensory epitheliain the inner ear of humans and other mammals. Thus, a partial orcomplete hearing loss due to damage or destruction of the sensory cellsof the inner ear is generally irreversible. In this respect the sensoryepithelia of the inner ear fundamentally differ from other tissues,where necrotic cells can be rapidly replaced by the division ofsubstitute cells and their subsequent maturation.

It is of interest that in other vertebrate classes, such as e.g. birds,necrotic sensory cells in the inner ear can be regenerated, unlike thesituation with humans. In birds sensory cells which have died afterdamage are replaced by so-called supporting cells located in theepithelium below the sensory cells. This takes place by division of thesupporting cells and subsequent maturation, a new supporting cell and asensory cell resulting from a supporting cell.

The discovery of the regeneration of sensory cells in the cochlea of thebird has over the past few years led to an attempt being made totransfer research results made on the bird to mammals and thereforeultimately humans. This inter alia promised success, because the cochleaof the bird and the cochlea of mammals have cell-biological points incommon. Both the sensory epithelium of bird cochlea and the sensorepithelium of mammal cochlea are postmitotic, i.e. sensory cells presentin the sensory epithelia are formed only during a specific time periodof embryonic development, after which normally no further cell divisionsoccur. However, this fundamental point in common makes it difficult tounderstand the phenomenon that in the vestibular sensory epithelium ofthe bird cell divisions can be detected throughout its life, but not inhumans.

As it was recognized in the bird that so-called growth factors can giverise to an increased proliferation rate in the bird cochlea, such growthfactors were also used in the mammal cochlea. However, it was notpossible to prove a reproducible action. This makes it obvious to drawthe conclusion that despite fundamental cell-biological points incommon, there must be other significant differences between bird andmammal cochlea. These could be that the supporting cells of the birdcochlea, as in the mammal, are postmitotic, but have only temporarilyleft the cell cycle. They can then reenter the cell cycle when acorresponding signal appears. Such supporting cells can be calledquiescent, i.e. they are in the waiting state. As opposed to this thesupporting cells of the mammal pass through a very high and specificdifferentiation and consequently irreversibly leave the cell cycle. Theycan consequently be called terminally differentiated and are e.g.comparable with neurons. This can apply in the case of the supportingcells of the mammal, which are referred to as so-called Pillar's orDeiter's cells. Such explanation models for cell-biological differencesbetween bird and mammal cochlea have given rise to a more detailedinvestigation of the regeneration of the sensory cells in the bird inorder to subsequently transfer the results obtained to mammals.

However, the problem of the present invention is to find a new startingpoint for the treatment of disorders or diseases of the inner ear, whichare linked with damage or destruction of the sensory cells of the innerear. The aim is less to transfer to mammals and in particular humansresults obtained on vertebrates other than mammals and more to makeavailable an action mechanism and corresponding active ingredients,which act directly in the cellular processes in the mammal andultimately lead to a regeneration of the sensory cells of the inner ear.

According to the invention, the process is characterized in that atleast one so-called cell cycle inhibitor present in the inner ear hasits inhibiting action partly inhibited or eliminated by at least oneactive ingredient, which results in a regeneration of the sensory cellsof the inner ear. From the patent law sense this process alsoincorporates the use of an active ingredient able to inhibit oreliminate the action of a cell cycle inhibitor present in the inner ear,either directly for the treatment of diseases or disorders of the innerear or indirectly for preparing a pharmaceutical composition or amedicament for the treatment of diseases or disorders of the inner ear,said diseases/disorders being linked with damage or destruction of thesensory cells of the inner ear.

The regeneration of the sensory cells of the inner ear resulting fromthe process according to the invention preferably takes place through astimulation of the proliferation of the supporting cells of the innerear, i.e. the supporting cells also present in the sensory epitheliumand usually located between and below the sensory cells. As there areone or more cell cycle inhibitors in the supporting cells of the innerear, by inhibiting or eliminating their inhibiting action by a suitableactive ingredient it is possible to initiate the cell division of thesupporting cells, thereby creating a fundamental prerequisite forcreating replacement or substitute cells for the necrotic or deadsensory cells. The cells resulting from the division of the supportingcells can then at least partly mature to functional sensory cells.

With regards to the sensory cells of the inner ear referred to up tonow, these are preferably so-called hair sensory cells or short haircells, which have at their upper end hair-like extensions, so-calledstereocilia or small sensory hairs. These hair cells are located on thebasilar membrane in the so-called corti-organ and form as so-calledouter and inner hair cells the actual receptor cells for acoustictransduction in the inner ear. Both the inner and the outer hair cellsare of interest for regeneration, regeneration of the outer hair cellsrepresenting a particular field of use of the invention as a result oftheir greater sensitivity. Those supporting cells which are anatomicallyparticularly well associated with the inner or outer hair sensory cellscan in particular be used for the active ingredient employed accordingto the invention. Thus, apart from outer hair sensory cells assupporting cells can be used the so-called Hensen's cells and, below theouter hair sensory cells, the so-called Deiter's cells and “outer”Pillar's cells. These Hensen's, Deiter's and outer Pillar's cells areconsequently particularly suitable as replacement cells for the outerhair sensory cells. Correspondingly alongside and below the inner hairsensory cells are provided the so-called inner sulcus cells assupporting cells and within the inner hair sensory cells also the innerPillar's cells, both being usable as replacement cells for the innerhair sensory cells. Thus, optionally a regeneration of inner or outerhair cells can be selectively initiated and influenced. Reference can bemade in this connection to the relevant textbooks and articlesconcerning the hearing process in mammals, particularly humans. Theregeneration of the hair sensory cells participating in acoustictransduction in the inner ear for the treatment of perceptive deafnessin the case of damage to said sensory cells represents the main field ofuse of the present invention.

The cell cycle inhibitors, whose inhibiting action is to be inhibited oreliminated according to the invention, can fundamentally be differentphysiological substances, particularly proteins, preventing the cellpassing through the normal cell cycle, including cell division. They arepreferably so-called cyclin-dependent kinase inhibitors (CDKIs). It isknown that during the development of an organism they are expressed to areinforced extent during the occurrence of terminally differentiatedcells and in this way prevent the reentry of the cell into the cellcycle. This would also explain the loss of the dividability of suchcells with reinforced expression of cyclin-dependent kinase inhibitors.Cell cycle inhibitors and in particular cyclin-dependent kinaseinhibitors of the so-called CIP/KIP family can be selectively expressedin specific cell types. Preferred cyclin-dependent kinase inhibitors arein particular the proteins referred to as p21^(Cip1), p27^(Kip1) andp57^(Kip2). According to the invention preference is given to thecyclin-dependent kinase inhibitor p27^(Kip1). As a result of theselective expression of such inhibitors and the different expressionpatterns resulting therefrom, the invention can be used for selectivelyinfluencing the cell cycle in a specific cell type. If e.g. in aspecific cell type, such as e.g. the supporting cells in the sensoryepithelium of the inner ear, p27^(Kip1) is expressed selectively or atleast with a significant proportion, by means of an active ingredientaimed specifically at this inhibitor, it is possible to eliminate itsinhibiting action and consequently initiate or stimulate theproliferation of supporting cells. By means of a maturation of at leastpart of the cells resulting from the division of the supporting cells, aregeneration of the sensory cells is brought about.

As is apparent from the statements up to now, according to the inventionthe inner ear disease or disorder involved is in particular a so-calledperceptive deafness. This is linked with the already described damage ordestruction of the hair sensory cells in the inner ear.

In the case of the active ingredient usable according to the invention,which inhibits or eliminates the inhibiting action of the cell cycleinhibitor, is preferably a substance, which normally acts inintracellular manner either directly or indirectly on the inhibitor,i.e. normally a peptide or protein. The active ingredient is preferablypresent in the form of a peptide or protein, which effects apeptide—peptide or protein—protein interaction with the inhibitor. Thiswould then be the case of a “direct” influencing of the function of theinhibitor. If the active ingredient is constituted by a nucleic acidmolecule, which codes one of the aforementioned peptides/proteins forthe amino acid sequence, it is possible to refer to an “indirect”influencing, because initially the coding nucleic acid molecule isintroduced into the corresponding cell and subsequently thepeptide/protein molecule (serving directly as the active ingredient) isexpressed. Said nucleic acid molecule can in particular be a recombinednucleic acid molecule. The nucleic acid molecule can fundamentally be aDNA molecule, a cDNA molecule or a RNA molecule.

Another active ingredient usable in preferred manner according to theinvention is a nucleic acid molecule, where use is made of the so-calledantisense method. In this method which is fundamentally known to theexpert use is normally made of a RNA, which is complimentary to the RNAof the normal (physiological) gene. This complimentary RNA is calledantisense-RNA. The antisense-RNA can prevent the synthesis of theprotein product belonging to the gene. In the case of the invention thismeans that a nucleic acid molecule, e.g. the antisense-RNA itself orDNA, during whose transcription the antisense-RNA is formed, isintroduced into the organism or cell for inhibiting or eliminating theinhibiting action of the cell cycle inhibitor. This introductionpreferably takes place with the aid of lipid compounds, which also carryviral components for the better docking and penetration of the nucleicacid molecule into the cell.

As stated, the active ingredient in the case of the invention can effecta direct interaction, preferably a peptide—peptide or protein—proteininteraction with the cell cycle inhibitor. However, the activeingredient can also indirectly inhibit or eliminate the inhibitingaction of the cell cycle inhibitor, in that it interacts at least aswell or preferably better with a physiological interaction partner ofthe cell cycle inhibitor than the cell cycle inhibitor itself. Thisprevents the cell cycle inhibitor from evolving its physiological(inhibiting) action.

Thus, e.g. in the case of the cyclin-dependent kinase inhibitorp27^(Kip1), it is known that it forms a protein complex together withthe cyclin-dependent kinase CDK2 and cyclin A. There are specific pointsat which peptide—peptide interactions occur between the p27^(Kip1) andthe CDX2 or cyclin A. Thus, e.g. identification has taken place of abonding point of very high affinity between p27^(Kip1) and cyclin A andseveral less strong bonding points between p27^(Kip1) and cyclin A orp27^(Kip1) and CDK2. On extracting one of the bonding points where thereis no high or very high affinity bonding/interaction, an activeingredient, preferably in the form of a further peptide/protein can beselected or developed can effect a bonding/interaction of at least ashigh or preferably higher affinity with one of the two interactionpartners at the particular bonding point. This inhibits or prevents thestandard physiological interaction, because the corresponding bondingpoint for the physiological interaction partner is blocked.

Thus, e.g. for a bonding point between p27^(Kip1) and cyclin A, but alsoCDK2, an optimized peptide structure or optimized amino acid sequencecan be developed for the amino acid sequence of p27^(Kip1) at thispoint, which then bonds with a better, i.e. higher affinity with thecorresponding sequence of cyclin A or CDK2 at this point. Such anoptimized peptide structure e.g. and preferably comprises up to 15 aminoacids and can then be directly introduced into the cell or preferablyexpressed in intracellular manner by an artificially introduced gene.Through the high affinity of such a peptide the interaction of thephysiological peptide partner is then destroyed and the formation of thepeptide complex, based on the inhibiting action of the cell cycleinhibitor is prevented. Thus, the active ingredient ensures an at leastpartial inhibition or a complete elimination of the inhibiting action ofthe cell cycle inhibitor. As a result of this process starting point ofthe invention the concentration of the active ingredient, particularlythe peptide/protein with the corresponding amino acid sequence in thecell only has to be roughly of the same level as the correspondingconcentration of the cell cycle inhibitor, whose action is to beinhibited or eliminated. As such concentrations, e.g. of p27^(Kip1) areapproximately 10 nM/l and roughly correspond to 1,000 to 10,000molecules per cell, even very low concentrations can suffice for theperformance of the invention. It is also important that for achievingsuch a concentration using gene-therapeutic methods it is sufficient tointroduce only a single copy of a DNA, coding for the correspondingamino acid sequence, for each cell. Compared with other methods whichhave to use much higher concentrations or a larger number of DNA copies,this represents a decisive advantage.

According to a further development the process according to theinvention can be performed in such a way that the active ingredient isin the form of a so-called vector or vehicle, said vector or vehiclecarrying at least one of the above-described nucleic acid molecules.Preferably it is a nucleic acid molecule, which codes for the amino acidsequence of a peptide or protein serving as the active ingredient. Saidvectors can be conventional viral and non-viral vectors, as are known tothe expert. When using viral vectors use can be made of retroviruses,adenoviruses or adeno-associated viruses. In the case of non-viralvectors it is known that no viral DNA participates, so that herefundamentally a “bare” DNA can be introduced into a cell. However,preferably such nucleic acid molecules are packed in so-called liposomesor lipoplexes and are introduced in this form into the organism andcell. The use of non-viral vectors or lipoplexes is fundamentallypreferred, because viral vectors have certain disadvantages known to theexpert. As a result of the above-described use possibilities of theinvention, it is here frequently possible to operate without using viralvectors, because the effectiveness of the active ingredients used isvery high and it is correspondingly possible to operate with lowconcentrations.

In the invention the active ingredient used is preferably employed in atherapeutically active quantity. In the conventional manner this can bematched to the subject undergoing treatment and inter alia use can bemade of known pharmaceutical additives. According to a furtherdevelopment the active ingredient used and correspondingly also theprocess according to the invention can be provided for localapplication. This makes it possible to avoid possible disadvantages of asystemic application. The target location of the process according tothe invention, namely the inner ear, is particularly suitable for localapplication. Thus, in the present case the active ingredient can beintroduced into the so-called perilymphatic space of the inner ear ofthe mammal, particularly human. This is a small liquid space with a veryslow exchange rate, which is accessible to therapeutic intervention fromthe middle ear, e.g. via the membrane of the circular window. Thisperilymphatic space has a volume of only approximately 20 microlitersand is also in direct contact with the cells of the corti-organ. Thisensures a direct action of the active ingredient on the sensoryepithelium with its hair cells and supporting cells.

The invention also relates to the actual active ingredient, whose use isdescribed in detail in the above-described process. This activeingredient is intended for the regeneration of the sensory cells of theinner ear, particularly the hair sensory cells of the inner ear and isable to at least partly inhibit or eliminate the inhibiting action of aso-called cell cycle inhibitor present in the inner ear. The cell cycleinhibitor is preferably a cyclin-dependent kinase inhibitor,particularly the cyclin-dependent kinase inhibitor p27^(Kip1). Referenceis made to the statements hereinbefore concerning the specific,preferred characteristics of the active ingredient. As stated, it can beat least one peptide/protein or at least one nucleic acid molecule, thelatter preferably being an antisense-DNA or antisense-RNA or preferablycodes for a corresponding peptide/protein usable as the activeingredient. The nucleic acid molecule can be a DNA molecule, a cDNAmolecule or a RNA molecule. In particular, the nucleic acid molecule isintroduced with the aid of a suitable vector or vehicle into theorganism or cell and these can be the described viral or non-viralvectors or nucleic acid molecules packed in liposomes/lipoplexes.

The invention finally relates to a pharmaceutical composition ormedicament, which contains at least one active ingredient able toinhibit or eliminate the action of a cell cycle inhibitor present in theinner ear, in an active quantity, as well as conventionally apharmaceutically acceptable carrier or support.

The described and further features of the invention can be gathered fromthe following description of a preferred embodiment in conjunction withthe subclaims, the example and the drawing. The individual features canbe implemented-individually or in the form of subcombinations.

FIG. 1 is an electron micrograph of a cell in nuclear division in thesensory epithelium of the corti-organ of a so-called p27^(Kip1) knockoutmouse.

EXAMPLE

For the test use was made of a so-called p27^(Kip1) knockout mouse(p27-/-), a mouse lacking the gene for expressing the proteinp27^(Kip1). Thus, in such a mouse p27^(Kip1) cannot evolve per se itscell cycle-inhibiting action.

The corti-organ is removed from such a p27^(Kip1) knockout mouse on theseventh day after birth (postnatal day 7) and is prepared in the usualway for electron microscopic examination making it possible to see thesensory epithelium of the corti-organ.

The result of the electron microscopic examination is shown in FIG. 1.This electronic micrograph shows that a cell in nuclear division(mitosis), i.e. a mitotic cell is located between two left-hand, upperor right-hand, lower, inner hair cells, whereof the black borderednuclei are at the left-hand top (complete) and right-hand bottom(partial). Mitosis is clearly visible on the condensed chromatin, thedissolved nuclear membrane and the basal body. the inner hair cell topleft and the basal body are given English-language captions in thedrawing to facilitate understanding.

FIG. 1 clearly shows that the lack of the cell cycle inhibitorp27^(Kip1) leads to the possibility of a cell division of supportingcells located there in the sensory epithelium of the corti-organ of themouse. Mention is also made of the fact that in the case of the celldivision shown in FIG. 1 it is not a single phenomenon within thesensory epithelium of the corti-organ, but instead a large number of thecells there undergo a cell division and therefore pass through the cellcycle. The phenomenon shown in FIG. 1 enables the conclusion to be drawnthat not only a cell division, but also following a cell division, whichrepresents the decisive step in the hair cell regeneration process,there is also a differentiation or maturation to hair sensory cells andfinally a functional recovery of the auditory function of the sensoryorgan. Thus, a regeneration of the sensory cells is possible. Thisconclusion is supported by the fact that in the case of the knockoutmouse there is not a single mitosis, but instead such knockout mice havemore hair cells than normal mice, in which the protein p27^(Kip1) isexpressed. Thus, the mitosis of the supporting cells also results inmatured sensory cells. The correctness of this conclusion is confirmedby the following results. In the case of heterozygous knockout mice theregeneration of hair cells was proved in that in the second week ofliving of the animals when they evolve the auditory function, the haircells were destroyed by the systemic administration of amikacin. After afurther two weeks without any injection the animals were killed andtheir cochlea examined. This revealed regenerated hair cells in thecochlea, which are marked or labelled by a proliferation marker or label(bromodesoxyuridine—BrdU) e.g. administered with the amikacin.

Thus, not only in knockout mice where the gene for p27^(Kip1) wasmissing from the outset, but also by inhibiting or eliminating thep27^(Kip1) expressed in the normal organism, e.g. with the aid of apeptide interacting with p27^(Kip1) or one of its physiologicalpartners, with the aid of the nucleic acid sequence coding for thispeptide or with the aid of an antisense-DNA/antisense-RNA it is possibleto bring about a regeneration of the sensory cells. This can also takeplace by an only partial elimination of the function of p27^(Kip1),because in the case of heterozygous mice a gene dose-dependent effect isobserved.

1. A process for the treatment of hearing loss caused by damaged innerear sensory hair cells, the process comprising the step of at leastpartly inhibiting or eliminating the action of p27^(Kip1) present in theinner ear by local administration of antisense molecules to mammalianp27^(Kip1) to the inner ear, thereby promoting regeneration of thesensory hair cells of the inner ear.
 2. The process according to claim1, characterized in that the regeneration of the sensory-hair cells ofthe inner ear takes place by stimulating proliferation of the supportingcells of the inner ear.
 3. A process for promoting regeneration andgrowth of sensory hair cells in the inner ear of a mammalian subject inneed thereof, the process comprising the step of locally administeringantisense molecules to mammalian p27^(Kip1) to the inner ear in anamount sufficient to promote regeneration and growth of sensory haircells in the inner ear.